WO2025128527A1 - Chemistry analytical rotor for bovine health management - Google Patents

Abstract

A method includes incubating a biological sample including a quantity of non-esterified fatty acids ("NEFA") with a reagent that includes a first component, a second component, and a third component. The first component is allowed to interact with the quantity of NEFA to generate a first reaction product. The first reaction product is allowed to interact with the second component to generate a second reaction product, and the second reaction product is allowed to interact with the third component to generate an indicator signal indicative of the quantity of NEFA. The indicator signal is interpreted as a determination of a quantity of the NEFA. The first reaction product, the second reaction product, and the indicator signal are generated in a single incubation operation. The reagent can include a first and a second reagent bead. The single incubation step can be performed in a rotor system.

Description

CHEMISTRY ANALYTICAL ROTOR FOR BOVINE HEALTH MANAGEMENT

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application 63/609,248, filed on December 12, 2023, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to chemical analytical rotor and uses thereof. Specifically, the invention relates to chemical analytical rotor for bovine health management.

BACKGROUND OF THE INVENTION

[0003] Monitoring and maintaining the health of a herd of cattle can be challenging. As a prey species, diseased cattle may not exhibit overt clinical symptoms for many days, which may prolong diagnosis and successful treatment. It would be beneficial to herd managers, owners, and veterinarians to have point-of-care tools to be able to monitor and maintain the health, welfare, and productivity of their herd of cattle. Point-of-care tools may allow the herd managers, owners, and veterinarians the ability to screen symptomatic and asymptomatic cattle receiving same day results which may help make critical health decisions to improve the health of the cattle and prevent economic loss. Most assays configured for detection of key analytes (e.g., non-esterified fatty acid “NEFA”) in a sample taken from cattle and useful for making critical health decisions for the cow may require processing of the sample and multiple incubation steps, events that can take valuable time and may not be able to be done on property. Disclosed herein is an analytic rotor, assays for detecting key analytes in a sample, and method of use that address the foregoing.

SUMMARY OF THE INVENTION

[0004] Disclosed herein is a method for detecting non-esterified fatty assay within a sample. In some embodiments, the method includes incubating a biological sample including a quantity of non-esterified fatty acids (“NEFA”) with a reagent, the reagent including a first component, a second component, and a third component, allowing the first component to interact with the quantity of NEFA to generate a first reaction product, allowing the first reaction product to interact with the second component to generate a second reaction product, allowing the second reaction product to interact with the third component to generate an indicator signal indicative of the quantity of NEFA, and interpreting the indicator signal to determine a quantity of the NEFA, wherein the first reaction product, the second reaction product, and the indicator signal are generated in a single incubation operation.

[0005] In some embodiments, the reagent comprises a first reagent bead and a second reagent bead.

[0006] In some embodiments, the first component comprises co-enzyme A, adenosine 5- triphosphate di sodium salt (“ATP”), and a acyl-coA synthase, and the first reaction product includes Acyl-CoA, adenosine monophosphate, and phosphoric acid (“PPi”).

[0007] In some embodiments, the first component is included in the first reagent bead.

[0008] In some embodiments, the second component includes acyl-CoA oxidase, and the second reaction product includes 2,3 -trans-enoyl-CoA and H2O2.

[0009] In some embodiments, the second component is included in the second reagent bead.

[0010] In some embodiments, third component includes an indicator molecule, 4- aminoantopyrine (4-AAP), and peroxidase.

[0011] In some embodiments, the indicator molecule is included in the first reagent bead, and the 4-AAP and peroxidase are included in the second reagent bead.

[0012] In some embodiments, the indicator molecule includes 2,4,6,-tribromo-3- hydroxybenzoic acid (“TBHBA”).

[0013] In some embodiments, the incubating is performed in a first reaction cuvette included in a rotor system. [0014] Also disclosed herein is a method, comprising (a) mixing a biological sample including a quantity of non-esterified fatty acids (“NEFA”) with a first reagent bead and a second reagent bead in a solution to form a mixture, the first reagent bead comprising a first enzyme, a second enzyme, a small molecule substrate, and a first chromogen and the second reagent bead comprises comprising a second chromogen different from the first chromogen, and a third enzyme different from any of the first enzyme, (b) incubating the mixture for a predetermined time at a predetermined temperature, (c) exposing the sample to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum, and (d) measuring an optical signal from the sample, the optical signal indicative of the quantity of NEFA in the biological sample.

[0015] In some embodiments, the first reagent bead further includes a salt (MgCk).

[0016] In some embodiments, the optical signal is reflectance, an absorbance spectrum, scattering spectrum, or an emission spectrum.

[0017] In some embodiments, at least steps (a) and (b) are performed in a reaction cuvette included in a rotor system.

[0018] In some embodiments, the sample is whole blood or plasma.

[0019] Also disclosed herein is a method for diagnosing the risk of metabolic disorder in a bovine animal, comprising collecting a biological sample from the bovine animal, assaying the sample for non-esterified fatty acids (“NEFA”), wherein the step of assaying comprises three reaction steps of a NEFA assay that occur in a single incubation operation and generate an indicator signal, and measuring an indicator signal to determine a concentration of the NEFA in the biological sample.

[0020] In some embodiments, the biological sample includes whole blood or plasma.

[0021] In some embodiments, the steps of assaying are performed in a reaction cuvette included in a rotor system.

[0022] Also disclosed herein is an assay configured to detect non-esterified fatty acids (NEFA) within a sample acquired from a bovine animal, comprising a composition comprising a first reagent bead comprising a first enzyme, a second enzyme, and a small molecule substrate, and a first chromogen, and a second reagent bead comprising a second chromogen different from the first chromogen, and a third enzyme different from any of the first enzyme, the second enzyme and third enzyme, the first bead and second bead configured to be mixed with the sample in a single incubation step.

[0023] In some embodiments, the assay occurs at a pH between 6.0 and 8.5 In some embodiments, the sample is whole blood or is plasma. In some embodiments, a total assay volume is about 100 pL.

[0024] In some embodiments, the first chromogen is 2, 4, 6-Tribromo-3 -hydroxy benzoic acid (“TBHBA”). In some embodiments, the second chromogen is 4-amino-antipyrine (“4- AAP”).

[0025] In some embodiments, the single incubation step is a homogenous incubation step.

[0026] In some embodiments, the small molecule substrate is Coenzyme A and is present at a concentration in a range of about 0.1 mM to about 0.5 mM.

[0027] In some embodiments, the first enzyme is Acyl-CoA synthetase and is present at a concentration in a range of about 1.5 U/mL to about 2.5 U/mL.

[0028] In some embodiments, the second enzyme is Acyl-CoA oxidase and is present at a concentration in a range of about 6.0 U/mL to about 9.0 U/mL.

[0029] In some embodiments, the third enzyme is peroxidase and is present at a concentration in a range of about 20 U/mL to about 30 U/mL.

[0030] In some embodiments, each of the first reagent bead and second reagent bead further include one or more cryoprotectants. In some embodiments, the one or more cryoprotectants is a surfactant. In some embodiments, the first reagent bead or the second reagent bead is lyophilized.

[0031] Also disclosed herein is an apparatus for detecting bovine health, comprising a first assay formulated to detect non-esterified fatty acids (“NEFA”) in a biological sample obtained from a bovine animal in a single incubation step with two or more distinct reagent beads, and one or more additional assays. In some embodiments, the apparatus is a rotor system. [0032] In some embodiments, the one or more additional assays are configured to detect serum amyloid A (“SAA”) and/or configured to detect P-Hydroxybutyric acid (“BHBA”).

[0033] In some embodiments, the SAA assay includes a reagent bead, the reagent bead includes a first capture biomolecule coupled to a first particle, the first capture biomolecule configured to bind to a first epitope of SSA, and a second capture biomolecule couples a second particle, the second capture biomolecule configured to bind to a second epitope of SSA, the first epitope of SAA being different from the second epitope of SAA.

[0034] In some embodiments, the first particle and the second particle include latex beads, the first and second particles configured to aggregate upon binding to the first and second epitopes of the SSA, respectively, and the aggregation of the first and second particles configured to cause a change in optical absorbance of the SAA assay, the change in optical absorbance indicative of a concentration of SAA in the biological sample.

[0035] In some embodiments, the BHBA assay includes one or more reagent beads, the one or more reagent beads including, nicotine adenine dinucleotide (NAD) in a concentration of about 1 mmol to about 30 mmol, D-3 -hydroxybutyrate dehydrogenase (3- HBDH) in a range of about 3 U/ml to about 300 U/ml, Diaphorase in a concentration of about 10 U/mL to about 50 U/mL, and 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (IN ) in a range of about 1 mmol/L to about 5 mM/L.

[0036] In some embodiments, the first assay is within a first reaction cuvette. In some embodiments, the one or more additional assays are within one or more separate reaction cuvettes. In some embodiments, the one or more additional assays include turbidimetric immunoassays or colorimetric enzymatic reactions.

[0037] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] FIG. 1A depicts a flow chart of an exemplary method used to detect NEFA in a sample, according to an embodiment.

[0040] FIG. IB depicts a schematic of an apparatus used in the exemplary methods described herein, according to an embodiment.

[0041] FIG. 2 depicts a various reaction steps of an exemplary assay configured to detect NEFA in a sample.

[0042] FIG. 3A depicts reaction steps of a conventional NEFA assay that includes two separate incubation steps.

[0043] FIG. 3B depicts reaction steps of an exemplary NEFA assay of the disclosure having a single incubation step.

[0044] FIG. 4 depicts a graph of the measured kinetics of samples with various concentrations of NEFA using the exemplary NEFA assay of the disclosure.

[0045] FIG. 5A depicts a plot of correlation of measured values using NEFA positive bovine samples and contrived samples using the exemplary NEFA assay of the disclosure.

[0046] FIG. 5B depicts a table summarizing reagent bead configurations for use in a rotorbased system for performing the exemplary NEFA assays described herein.

[0047] FIG. 6 depicts a graph of measured icteric values for determining icteric interference for the exemplary NEFA assay of the disclosure.

[0048] FIG. 7 depicts a graph of measured hemolysis values for determining hemolysis interference for the exemplary NEFA assay of the disclosure.

[0049] FIG. 8 depicts a graph of measured lipid values for determining lipid interference for exemplary NEFA assay of the disclosure. [0050] FIGS. 9A-9B depicts graphs of measured percent recovery of NEFA after days 0, 3, and 10 for determining the accelerated stability of the exemplary NEFA assay of the disclosure at 35°C.

[0051] FIG. 9C depicts a table of the measured percent recovery of NEFA after day 3 and day 10 for determining the accelerated stability for the exemplary NEFA assay of the disclosure.

[0052] FIG. 10 depicts a graph of the linear dynamic range of the exemplary NEFA assay of the disclosure.

[0053] FIG. 11 depicts a table of linear fit for the exemplary NEFA assay of the disclosure.

[0054] FIG. 12 depicts a schematic of an illustration of an immunoturbidimetric reaction used in an exemplary assay to detect SAA.

[0055] FIG. 13A depicts a graph of the linearity of SAA calibrator samples as measured by the exemplary SAA assay of the disclosure.

[0056] FIG. 13B depicts a graph of measured values that show the correlation between the measured and reference method-assigned values for SAA positive bovine samples using the exemplary SAA assay of the disclosure.

[0057] FIG. 13C depicts a graph of measured hemolysis values for determining hemolysis interference for the exemplary SAA assay of the disclosure.

[0058] FIG. 13D depicts a graph of measured lipid values for determining lipid interference for exemplary SAA assay of the disclosure.

[0059] FIG. 13E depicts a graph of measured icteric values for determining icteric interference for the exemplary NEFA assay of the disclosure.

[0060] FIG. 14 depicts a schematic of a reaction of an exemplary assay used to detect BHBA, according to an embodiment.

[0061] FIG. 15A depicts a graph of assay Linearity with measured values compared to reference method values for the same samples for determining the exemplary assay endpoints for the exemplary assay used to detect BHBA of the disclosure. [0062] FIG. 15B depicts a graph of measured kinetic values for samples having various concentrations of BHBA using the exemplary BHBA assay of the disclosure.

[0063] FIG. 16 depicts a graph of measured values that depict the correlation between the measured and reference method-assigned values for BHBA positive bovine samples using the exemplary BHBA assay of the disclosure.

[0064] FIG. 17 depicts a graph of the accelerated stability of the exemplary BHBA assay of the disclosure. Specifically, it depicts measured percent recovery of BHBA samples measured at day 0 and after days 3, 10, 14, 21 of theassay reagents being stored at a 35°C with four different rotor lots.

[0065] FIG. 18 depicts a graph of measured hemolysis values for determining hemolysis interference in the exemplary BHBA assay of the disclosure.

[0066] FIG. 19 depicts a graph of measured lipid values for determining lipid interference in the exemplary BHBA assay of the disclosure.

[0067] FIG. 20 depicts a graph of measured icteric values for determining icteric interference in the exemplary BHBA assay of the disclosure.

[0068] FIG. 21 depicts a flow chart of an exemplary method of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0069] Embodiments described herein are related to a rotor-based system to detect one or more analytes within a sample. Specifically, embodiments described herein may include one or more exemplary assays that may be used to detect one or more analytes within a sample taken from an animal including, in some embodiments, a bovine animal. The one or more exemplary assays may include assays to detect non-esterified fatty acid (“NEFA”), serum amyloid A (“SAA”), or -hydroxybutyric acid (“BHBA”).

[0070] NEFA, SAA, and BHBA are important indicators of a bovine animal’s health. Monitoring these parameters is desirable for herd health assessment, prognostic indication, and monitoring health of bovine animals, for example, health of transition dairy cattle. The transition period is the time in which dairy cattle undergo a large metabolic requirement change which makes managing herd health critical to protect its value, while ensuring a high return on investment. NEFA assay principle is based on the formation of a highly stable dye by an oxidation coupling reaction. Conventional NEFA assays for bovine animal health include multiple reaction steps, that are performed in multiple discrete incubation steps. For example, the reaction steps of conventional NEFA assays can be carried out only in multiple independent steps requiring more effort and time. All assay kits follow heterogenous incubation steps. The reagent handling in multiple steps can cause serious eye damage. Conventional NEFA assays generally require the whole blood to be processed before testing, for example, to obtain plasma from the blood for performing the assay. Large volumes of blood are generally extracted in such conventional NEFA assays to generate enough plasma for performing the assay. This makes conventional NEFA assays expensive, increases the manpower for performing such assays, increases complexity and hence, skill needed to perform such assays, and increases turnaround times. Moreover, there is no diagnostic assay available that can monitor NEFA, SAA, and BHBA in a single platform.

[0071] In contrast, systems and methods described herein for detecting NEFA, SAA, and/or BHBA provide one or more advantages including, for example: (1) enabling detection of NEFA in a single incubation step, thereby reducing cost, complexity, and assay time; (2) allowing monitoring of NEFA, SAA, and BHBA in a single platform, thus further reducing health monitoring time and cost; (3) allowing assay to be performed using whole blood without having to process the blood, thus reducing sample volume and time for performing the assay; (4) providing herd managers, owners and veterinarians with a point- of-care, on farm tool to screen asymptomatic cattle to achieve same day results, allowing the producers, veterinarians, and herd managers to make critical health decisions prior to the cattle exhibiting clinical symptoms to both improve the health, welfare and prevent economic losses; and (5) allowing other diagnostic assays to be integrated with NEFA, SAA, and BHBA on the same platform such as, for example, Albumin (ALB), Calcium (CA), Magnesium (MG), Phosphorus (PHOS), Urea Nitrogen (BUN), and Potassium (K).

[0072] Disclosed herein is a method of detecting NEFA within a sample. FIG. 1 A depicts a flow chart of an exemplary method of detecting NEFA within a sample, according to an embodiment. In some embodiments, the method 100 may include providing a biological sample to a rotor-based system, wherein the biological sample includes an unknown quantity of NF. FA therein, at 102. The method 100 may further include incubating the biological sample with a reagent including a first component, a second component, and a third component, at 104. The method 100 further includes allowing the first component to interact with the unknown quantity of NEFA to generate a first reaction product, at 106. The method 100 further includes allowing the first reaction product to interact with the second component to generate a second reaction product, at 108. The method 100 further includes allowing the second reactant product to react with the third component to generate an indicator signal indicative of the quantity of NEFA within the sample, at 110. The method 100 further includes interpreting the indicator signal to determine the quantity of NEFA within the sample, at 112. Advantageously, steps 104-110 all take place within a single incubation step, reducing the time of the assay along with reagents of the assay. Until now, many assays used to detect NEFA within a sample would include a plurality of steps including multiple distinct incubation steps, prolonging both the assay and the time required until a decision may be made about the health of the subject from which the sample was taken. As described in more detail herein, this exemplary method of detecting NEFA within a test sample using a single incubation step as described below is novel and inventive.

[0073] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0074] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, the term “a component” is intended to mean a single component or a combination of components, “a material” is intended to mean one or more materials, or a combination thereof.

[0075] As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0076] The phrase “and/or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0077] As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.

[0078] As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements), etc.

[0079] In the embodiments, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0080] As used herein, the term “set” and “plurality” can refer to multiple features or a singular feature with multiple parts. For example, when referring to a set of electrodes, the set of electrodes can be considered as one electrode with multiple portions, or the set of electrodes can be considered as multiple, distinct electrodes. Additionally, for example, when referring to a plurality of electrochemical cells, the plurality of electrochemical cells can be considered as multiple, distinct electrochemical cells or as one electrochemical cell with multiple portions. Thus, a set of portions or a plurality of portions may include multiple portions that are either continuous or discontinuous from each other. A plurality of particles or a plurality of materials can also be fabricated from multiple items that are produced separately and are later joined together (e.g., via mixing, an adhesive, or any suitable method). Rotor-Based Systems

[0081] The exemplary assays disclosed herein used to detect one or more analytes within a sample may use rotor-based systems including rotor-based devices of the disclosure. Referring to FIG. IB, a schematic of rotor-based system is shown, according to an embodiment. The rotor-based device 200 may include a rotor 210 having one or more reaction chambers 212a-212c therein. In some embodiments, the one or more reaction chambers 212a-212c may include reaction cuvettes, reaction wells, reaction discs, reaction chips or the like. In some embodiments, each reaction chamber may include an exemplary assay of the disclosure. A sample containing an unknown amount of target analyte may be added to the device 200. For example, a portion of the sample may be disposed in and contained within each of the one or more reaction chambers 212a-212c.

[0082] In some embodiments, the rotor 210 may include a centrifugal rotor. The rotorbased device 200 may further include a light source 230 configured to emit light at a wavelength range within the ultraviolet-visible-infrared spectrum, and a detector 232 configured to detect light within the wavelength of ultraviolet-visible-infrared spectrum. As the rotor 210 rotates, the reaction chambers 212a-212c may pass through a light emitted by the light source 230. The contents of the reaction chamber may reflect, emit, or scatter the light emitted by the light source 230, which may be detected by the detector 232. The combination of the light source 230 and the detector 232 may allow the device 200 to measure optical signals including changes in optical signals from the sample. In some embodiments, the optical signal may include reflectance, an absorbance spectrum, a scattering spectrum, or an emission spectrum. For example, in some embodiments, the contents of the reaction chamber (e.g., the sample) may produce an enhanced shift in the peak absorption wavelength which is detectable by the detector 232 and may indicate the presence of the target analyte (e.g., NEFA, SAA, and/or BHBA) within the sample.

[0083] In some embodiments, the same exemplary assay may be within each of the reaction chambers 212a-212c, while in other embodiments, different exemplary assays may be within each reaction chambers 212a-212c. For example, in some embodiments, a first exemplary assay configured to detect a first target analyte may be contained within a first reaction chamber 212a, a second exemplary assay configured to detect a second target analyte may be contained within a second reaction chamber 212b, and a third exemplary assay configured to detect a third target analyte may be contained within a third reaction chamber 212c. Examples of rotor-based systems that may be used with the methods and assays described herein are described in US Patent No. 9,816,987, issued November 14, 2017, and entitled “Rotors for Immunoassays,” the entire disclosure of which is incorporated herein by reference, and attached hereto as Exhibit A.

[0084] In some embodiments, the rotor-based device 200 may include a sample port 234 wherein a sample may be contacted with the rotor 210. The application of a centrifugal force to the rotor may deliver the sample to each of the one or more reaction chambers 212a-212c, wherein the sample is mixed with the exemplary assay contained within the reaction chamber.

Exemplary Assays

[0085] In some embodiments, the exemplary assays used within the rotor-based systems (e.g., the system 200) described herein may be located within the one or more reaction chambers of the rotor-based system. Each chamber of the one or more reaction chambers may include detection conjugates coupled to binding partners, capture biomolecules capable of specifically binding to a target analyte, or reaction molecules capable of reacting with a target analyte within the sample. In some embodiments, each of the detection conjugates, capture biomolecules, or reaction molecules may include a lyophilized composition. In some embodiments, each of the detection conjugates, the capture biomolecules, or the reaction molecules may be immobilized on a surface of the reaction chamber. For example, in some embodiments, the reaction chamber may include a surface containing a metallic nanolayer. In some embodiments, the capture biomolecules may include antigens or antibodies, as will be described in more detail herein. In some embodiments, each of the reaction chambers of the rotor-based system may include one or more exemplary assays of the disclosure, wherein one or more samples from a single subject or from multiple subjects may be tested using the one or more assays of the disclosure. Sample Volumes and Reaction Time of the Exemplary Assays

[0086] In some embodiments, one or more of the exemplary assays of the disclosure may reduce the number of reaction steps or the number of incubation steps, advantageously, reducing the total reaction time of the assay. In some embodiments, the exemplary assays may have a specific total reaction time. The total reaction time may include the time from providing the sample to the rotor-based system to receiving a measurement of the target analyte within the sample. For example, in some embodiments, the total reaction time of the exemplary assays may be within the range of about 1 seconds to about 50 minutes including about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, about 90 seconds, about 95 seconds, about 100 seconds, about 105 seconds, about 110 seconds, about 115 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes, inclusive.

[0087] In some embodiments, one or more of the exemplary assays described herein may have improved specificity and/or improved sensitivity. Advantageously, improved sensitivity and/or improved specificity may allow some assays of the disclosure to be performed using smaller sample volumes, reducing the amount of sample needed to be acquired from a subject. In some embodiments, the exemplary assays may be performed using a specific sample volume. In some embodiments, the sample volume used within the exemplary assays of the disclosure may include 10 pL, 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL, 110 pL, 120 pL, 130 pL, 140 pL, 150 pL, 160 pL, 170 pL, 180 pL, 190 pL, or 200 pL, inclusive. [0088] In some embodiments, one or more of the exemplary assays may be performed using a specific total assay volume, wherein the total assay volume may include the total volume of the solution within the reaction chamber. For example, in some embodiments, the total assay volume may be within the range of about 25 pL to about 1000 pL, inclusive, for example, including about 25 pL, about 50 pL, about 75 pL, about 100 pL, about 125 pL, about 150 pL, about 175 pL, about 200 pL, about 225 pL, about 250 pL, about 275 pL, about 300 pL, about 325 pL, about 350 pL, about 375 pL, about 400 pL, about 425 pL, about 450 pL, about 475 pL, about 500 pL, about 525 pL, about 550 pL, about 575 pL, about 600 pL, about 625 pL, about 650 pL, about 675 pL, about 700 pL, about 725 pL, about 750 pL, about 775 pL, about 800 pL, about 825 pL, about 850 pL, about 875 pL, about 900 pL, about 925 pL, about 950 pL, about 975 pL, or about 1000 pL, inclusive.

[0089] In some embodiments, a single volume of sample may be delivered to the rotorbased system, which may direct a specific sample volume to each of the one or more reaction chambers. In some embodiments, in which the rotor-based system includes multiple reaction chambers and/or multiple assays, the rotor-based system may direct the same sample volume to each reaction chamber or may direct different sample volumes to each reaction chamber.

Samples

[0090] In some embodiments, the sample may include a biological sample. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, urine, pleural effusion, sweat, bile, cerebrospinal fluid, fecal material, vaginal fluids, sperm, ocular lens fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, biopsy tissues, saliva, and cellular lysates. In some embodiments, the biological sample may be obtained from a human subject, or an animal subject suspected of having a disease condition, such as cancer, infectious diseases (e.g., viral-, bacterial-, parasitic- or fungal-infections), cardiovascular disease, metabolic disease, autoimmune disease, etc. In some embodiments, the biological sample may also be obtained from a healthy subject (e.g., human or animal) undergoing a routine medical check-up. In some embodiments, the animal subject may include ruminant species including a bovine animal (e.g., a cow), a porcine animal (e.g., a pig), a caprine animal (e.g., a goat), an ovine animal (e.g., sheep), lamb, etc. [0091] In some embodiments, the exemplary assays of the disclosure may include detection conjugates, capture biomolecules, or reaction molecules that are structured as beads or reagent beads. In some embodiments, the beads may have suitable diameter ranges including from about 5 nm to about 200 nm, from about 10 nm to about 100 nm, and from about 20 nm to about 60 nm. In some embodiments, the beads may include a specific material or combinations of materials or reagents. For example, in some embodiments, the beads may include latex, polyvinyl, nylon, polyester, or the like. In some embodiments, the one or more beads described herein may be lyophilized. In some embodiments, the beads may include a single component or multiple components, as will be described in more detail herein.

Exemplary Assays

NEFA

[0092] In some embodiments, the one or more exemplary assays used in the rotor-based system may be an exemplary assay used to detect NEFA within a sample. Increased NEFA concentrations within a subject may be indicators of elevated post-partum disease risk in bovine animals and may be helpful in assessing dry cow nutritional management in conjunction with cholesterol determination. In some embodiments, elevated NEFA concentrations may be an indication of post-partum diseases including but not limited to ketosis, fatty liver, displaced abomasums, retained placentras, and metritis.

[0093] FIG. 2 depicts a schematic of a reaction that may be used with in the exemplary assay to detect NEFA within a sample. In some embodiments, the exemplary assay to detect NEFA within a sample of the disclosure may include three reaction steps that are all performed within a single incubation step and may occur simultaneously or subsequently. In the first reaction step (1), the unknown quantity of NEFA within the sample in the presence of coenzyme A (CoA) and adenosine 5 -triphosphate disodium salt (ATP) is converted to acyl-CoA, adenosine monophosphate (“AMP”) and pyrophosphoric acid (PPi) by the action of Acyl-CoA synthetase. In a second step (2), acyl-CoA generated in the first reaction step is oxidized by acyl-CoA oxidase, which results in 2,3-trans-enoyl-CoA and hydrogen peroxide by the action of acyl-CoA oxidase (“ACOD”). In the third reaction step (3), the hydrogen peroxide generated in the second reaction step, yields a colored pigment in the presence of peroxidase by quantitative oxidation condensation with 4- aminoantipyrine (“4-AAP”) and an indicator molecule. In some embodiments, the indicator molecule may include a chromogen including, but not limited to 2,4,6,-tribromo- 3 -hydroxybenzoic acid (“TBHBA”), (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m -toluidine, monosodium salt, f'TOOS”), 3-methy1-N-ethy1-N-(P-hydroxyethyl)-aniline (“MEHA”), or 4-aminoantipyrine (“4-AAP”). In some embodiments, the chromogen includes TBHBA. The exemplary assay for detection of NEFA within a sample is based on the detection principle of NEFA being converted to CoA products with the generation of H2O2, wherein the amount of H2O2 produced during the reaction can be directly correlated with the total NEFA concentration in the sample, due to the reaction of H2O2 with peroxidase.

[0094] In the exemplary assay to detect NEFA in a sample, an unknown quantity of NEFA within the sample may react with a first component, that may include coenzyme A along with adenosine 5 -triphosphate disodium salt (ATP) and acyl-CoA synthetase. This reaction may generate a first reaction product that may include acyl-CoA, AMP and PPi. The first reaction product may interact with a second component that may include acyl- CoA oxidase and dioxide. This reaction may produce a second reaction product that may include hydrogen peroxide along with 2,3-trans-enoyl-CoA. The second reaction product may react with a third component, that may include an indicator molecule, 4-AAP and peroxidase. In some embodiments, the indicator molecule may include a chromogen. In some embodiments, the reaction of the second reaction product with the third component may produce the indicator signal (e g., a color or a change in color) that may be detected at a specific wavelength, for example, using the rotor-based system described herein.

[0095] The entire reaction depicted within FIG. 2, in an assay, occurs in multiple steps. As previously described herein, in a conventional NEFA assay shown in FIG. 3A, multiple discrete incubation steps are performed to determine the quantity of NEFA within a target sample. For example, the unknown amount of NEFA within the sample may react with the first component to produce the first reaction product in a first incubation step (“Incubation 1”). This first reaction product may be separated using standard biochemical techniques for separating a first reaction product from a solution. This first reaction product may then be added to a second component and incubated in a second incubation step (“Incubation 2), where the first reaction product may react with the second component to produce the second reaction product. Within the same incubation step, the second reaction product may interact with a third component to produce the indicator signal. Since this reaction requires multiple incubation steps, it is often times performed in different reaction containers over a period of time. Moreover, the second reaction is generally performed with plasma rather than blood thus adding another step of separating plasma from blood in a conventional assay.

[0096] In contrast, in the exemplary assay of the disclosure used to detect NEFA within a target sample as seen in FIG. 3B, the entire reaction advantageously occurs in a single homogenous incubation step and may occur in a single reaction container. The unknown quantity of NEFA within the sample reacts with the first component to generate the first reaction product which reacts with the second component to generate a second reaction product, which reacts with the third component to generate the indicator signal that may be detected at a certain wavelength and quantified, indicative of the quantity of NEFA within the sample.

[0097] In order for the reaction to occur in a single homogenous incubation step and in a single reaction container, the exemplary assay may use two or more beads within a single reaction chamber, where the two or more beads include one or more reagents used within the reaction of FIG. 3B. In some embodiments, the two or more beads (“reagent beads”) provide sufficient concentrations of each of the first component, second component, and third component for the reaction to occur in the presence of an unknown concentration of NEFA. In some embodiments, the two or more beads may have different formulations, wherein the combination of the beads provide all the necessary reagents for the reaction to occur. It can be appreciated that the necessary reagents for the reactions to occur may be formulated into a first bead or a second bead in any number of combinations, all of which are considered in this application. In some embodiments, the two or more beads may each include different reagents. In some embodiments, the two or more beads may include about two beads, about three beads, about four beads, about five beads, about six beads, about seven beads, about eight beads, about nine beads, or about ten beads. In such embodiments, two or more of the beads may include the same reagent, or each of the two or more beads may include different reagents. [0098] For ease of the disclosure, a formulation for a first reagent bead and a formulation for a second reagent bead are discussed below.

[0099] In some embodiments in which the exemplary assay uses two beads, a first reagent bead may include the first component and a second reagent bead may include the second component. In some embodiments, any of the first reagent bead or second reagent bead may include one or more of the first component, second component, or the third component including any one of the following reagents: a first chromogen, a first enzyme, a second enzyme, a third enzyme, a second chromogen, a small molecule substrate, and one or more cryoprotectants. In some embodiments, each of the first enzyme, the second enzyme, or the third enzyme, may be different. In some embodiments, each of the first chromogen and the second chromogen may be the same or may be different chromogens.

[0100] In some embodiments, the first component may include the small molecule substrate. In some embodiments, the small molecule substrate may include coenzyme A. In some embodiments, the first reaction product may include Acyl-CoA, AMP and PPi. In some embodiments, the second component may include Acyl-CoA oxidase. In some embodiments, the second reaction product may include 2,3 -trans-enoyl-CoA and H2O2. In some embodiments, the third component may include an indicator molecule (e.g., a chromogen), 4-AAP, and peroxidase. In some embodiments, the first enzyme may include Acyl-CoA synthetase. In some embodiments, the second enzyme may include Acyl-CoA oxidase. In some embodiments, the third enzyme may include peroxidase.

[0101] In some embodiments, the one or more chromogens may include any chromogen selected from the group consisting of: ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, MAOS, TODB, TOPS, TOOS, MEHA, TBHBA, and 4-AAP.

[0102] In some embodiments, the one or more reagent beads may include additional reagents including, but not limited to buffers, adenosine phosphate (e.g., ATP), one or more cofactors, salts, zwitterionic detergent, a surfactant, water, one or more water soluble antibiotics, one or more antimicrobial agents, small molecule substrates or the like. In some embodiments, the buffers may include 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7). In some embodiments, the one or more cofactors may include salts including MgCh, orNaCl. In some embodiments, the additional reagents may include anions or salts that may reduce interference such as ferrocyanide or the like. In some embodiments, the zwitterionic detergent may include n-Tetradecyl-N,N-dimethyl-3-ammonio-l- propanesulfonate. In some embodiments, the surfactant may prevent most of the bivalent metal interference and may include polyethylene glycol (e.g., PEG3400). In some embodiments, the one or more cryoprotectants may include sugars, proteins, and surfactants. In some embodiments, after formation, the two or more reagent beads may be stored at about 2-8° C.

[0103] In some embodiments, the first reagent bead and/or the second reagent bead may include any formulation as laid out in tables below including Tables 1-8.

[0104] In some embodiments, the exemplary assay configured to detect NEFA within a sample may be configured to detect NEFA within a sample acquired from a bovine animal. In some embodiments, the exemplary assay may include a composition including the first reagent bead, and the second reagent bead, the first reagent bead and second reagent bead configured to be mixed with the sample and reacted therewith in a single incubation step. In some embodiments, the first reagent bead may include a first enzyme, a second enzyme, a first small molecule substrate, and a first chromogen. In some embodiments, the second reagent bead may include a second chromogen different from the first chromogen, and a third enzyme different from any of the first enzyme, or the second enzyme. In some embodiments, the exemplary assay may occur at a pH within the range of about 6.0 and about 8.5 including about 6.0, about 6.1, about 6.2 about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5, inclusive. In some embodiments, the single incubation step may include a homogenous incubation step. In some embodiments, each of the first reagent bead and the second reagent bead may further include one or more cryoprotectants as described above, wherein the one or more cryoprotectants may include a surfactant, one or more sugars, or one or more proteins. In some embodiments, the surfactant may include polyethylene glycol and may be present at the concentration of about 0.3 vol% to about 0.7 vol%, inclusive. [0105] In some embodiments, the first small molecule substrate may be coenzyme A and may be included within the one or more reagent beads at a concentration within the range of about 0.1 mM to about 0.5 mM including about O.lmM, about 0.2 mM, about 0.3 mM, about 0.4 mM, or about 0.5 mM. In some embodiments, the first enzyme may be Acyl- CoA synthetase and may be included within the one or more reagent beads at a concentration within the range of about 1.5 U/mL to about 2.5 U/mL including about 1.5 U/mL, about 1.6 U/mL, about 1.7 U/mL, about 1.8 U/mL, about 1.9 U/mL, about 2.0

U/mL, about 2.1 U/mL, about 2.2 U/mL, about 2.3 U/mL, about 2.4 U/mL, or about 2.5 U/mL, inclusive. In some embodiments, the second enzyme may be Acyl-CoA oxidase and may be included within the one or more reagent beads at a concentration within the range of about 6.0 U/mL to about 9.0 U/mL including about 6.0 U/mL, about 6.1 U/mL, about 6.2 U/mL, about 6.3 U/mL, about 6.4 U/mL, about 6.5 U/mL, about 6.6 U/mL, about 6.7

U/mL, about 6.8 U/mL, about 6.9 U/mL, about 7.0 U/mL, about 7.1 U/mL, about 7.2

U/mL, about 7.3 U/mL, about 7.4 U/mL, about 7.5 U/mL, about 7.6 U/mL, about 7.7

U/mL, about 7.8 U/mL, about 7.9 U/mL, about 8.0 U/mL, about 8.1 U/mL, about 8.2

U/mL, about 8.3 U/mL, about 8.4 U/mL, about 8.5 U/mL, about 8.6 U/mL, about 8.7

U/mL, about 8.8 U/mL, about 8.9 U/mL, or about 9.0 U/mL, inclusive. In some embodiments, the third enzyme may be peroxidase and may be included within the one or more reagent beads at a concentration within the range of about 20 U/mL to about 30 U/mL including about 20 U/mL, about 21 U/mL, about 22 U/mL, about 23 U/mL, about 24 U/mL, about 25 U/mL, about 26 U/mL, about 27 U/mL, about 28 U/mL, about 29 U/mL, or about 30 U/mL, inclusive.

[0106] In some embodiments, the first reagent bead may include the formulation of MOPS (pH 7) with a final concentration of about 44.21 ImM, ATP with a final concentration of 2.21 ImM, MgCb with a final concentration of 3.316mM, TBHBA with a final concentration of 4.421mM, Coenzyme A with a final concentration of 0.387mM, Acyl- CoA synthetase with a final concentration of 2.211 U/mL, Acyl-CoA oxidase with a final concentration of 8.842 U/mL, one or more cryoprotectants with a final concentration of 1 .736%, a Zwitterionic detergent with a final concentration of 0.026%, and water.

[0107] In some embodiments, the second reagent bead may include the formulation of MOPS (pH 7) with a final concentration of 52.500mM, 4-AAP with a final concentration of 1 ,547mM, Ferrocyanide with a final concentration of 0.22 ImM, Peroxidase with a final concentration of 26.316 U/mL, one or more cryoprotectants with a final concentration of 1.736%, polyethylene glycol with a final concentration of 0.526%, and water.

Serum Amyloid A Assay

[0108] Another exemplary assay of the disclosure that may be used, for example, within the rotor-based system, to detect one or more analytes within a sample may include an assay configured to detect Serum Amyloid A (“SAA”). SAA is an apolipoprotein that may be associated with high-density lipoprotein (“HDL”). SAA may be an inflammatory marker. In some subjects (e.g., cows), the presence of SAA within a sample or elevated levels of SAA may indicate the presence of inflammatory diseases within a subject that need to be further investigated.

[0109] FIG. 12 depicts a schematic of a reaction used within the exemplary assay to detect an unknown quantity of SAA within a sample. The exemplary assay uses immunoturbidimetry including turbidimetric agglutination to determine the concentration of SAA within a sample. Within the reaction, generally, a first capture biomolecule (e.g., first antibody “Antibody 1”) and a second capture biomolecule (e.g., second antibody “Antibody 2”) may be conjugated to the surface of detection conjugates to form antibodyconjugates. The first antibody and second antibody may bind to the same epitope of SAA or different epitopes of SAA, for example, separate from and ideally non-overlapping with the epitope to which the first antibody binds the target analyte. The antibody-conjugates are mixed with the unknown quantity of SAA within a solution including a sample. If SAA is present within the sample, the antibody-conjugates will bind to SAA and agglutination will occur. Agglutination within the solution will change the absorbance, which can be measured as a function of time to determine the concentration of SAA.

[0110] Assays that use one or more antibodies to detect SAA may be using antibodies configured to detect SAA within both humans and animals. For example, these antibodies are developed from partial bovine sequences and the human genome. These antibodies may have reduced specificity for bovine SAA compared to antibodies raised to detect SAA solely in bovine, due to amino acid differences in SAA in humans or animals. As disclosed herein, the antibodies used within the exemplary assay to detect SAA may be antibodies raised specifically against bovine SAA, using bovine genetic sequences. This would lead to an improvement of the sensitivity and the specificity towards bovine species in the assay. For example, the increased specificity of antibody may allow for a reduction in the concentration of antibodies used within the assay and/or a greater range of detection of SAA within a sample. In some embodiments, the antibodies used within the exemplary SAA assay of the disclosure may include monoclonal antibodies. In some embodiments, a first antibody may be configured to bind to a first epitope of SAA and a second antibody may be configured to bind to a second epitope of SAA, wherein upon binding to SAA aggregation is promoted.

[0111] In some embodiments, the first antibody or the second antibody may bind to the protein comprising an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% sequence identity thereto.

MKLFTGLILCSLVLGVHSQWMSFFGEAYEGAKDMWRAYSDMREANYKGADKY FHARGNYDAAQRGPGGAWAAKVISDARENIQRFTDPLFKGTTSGQGQEDSRAD QAANEWGRSGKDPNHFRPAGLPDKY (SEQ ID NO: 1)

[0112] In some embodiments, the first epitope and second epitope may be within the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% sequence identity thereto.

[0113] The exemplary assay used to detect SAA within a sample may also be used within the context of the rotor-based system. The exemplary assay may use beads as detection conjugates, wherein the monoclonal antibodies of the disclosure are conjugated to the surface of the beads. In some embodiments, the antibodies may be passively adsorbed onto the surface of the beads. In some embodiments, the beads may include latex beads. The latex beads (e.g., reagent bead) may include one or more reagents, include one or more buffers, a latex bead mixture, one or more monoclonal antibodies, one or more sugars, one or more proteins, and one or more salts. In some embodiments, the one or more buffers may include HEPES. In some embodiments, HEPES may be present at a percent composition of about 50-250 mM including about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, or about 250 mM, inclusive. In some embodiments, the first antibody and second antibody may be present at a volume percent composition of about 0.01 % to about 0.05% including about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%, inclusive.

[0114] In some embodiments, the one or more sugars include trehalose. In some embodiments, trehalose may be present at a volume percent composition of about 3% to about 5% including about 3%, about 3.2%, about 3.4%, about 3.6%, about 3.8%, about 4.0%, about 4.2%, about 4.4%, about 4.6%, about 4.8%, or about 5.0%, inclusive. In some embodiments, the one or more proteins may include bovine serum albumin (BSA). In some embodiments, the BSA may be present at a percent composition of about 1% to about 3% including about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%, inclusive. In some embodiments, the one or more salts may include NaCl. In some embodiments, NaCl may be present at a percent composition of about 0.5M to about 1.0M including about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, or about 1.0M, inclusive.

[0115] In some embodiments, the SAA reagent bead may comprise a formulation of Table 1.

Table 1: SAA Reagent Bead Formulation

B-Hydroxybutyric acid Assay (BHBA)

[0116] In some embodiments, another one or more exemplary assays used in the rotorbased system may be an exemplary assay used to detect BHBA within a sample. During times of elevated NEFA uptake within a subject (e.g., cow), some NEFA may be converted back to triglycerides and stored in the liver of the subject. The triglycerides may be incompletely oxidized to ketones such as BHBA. Subjects (e.g., cows) with high BHBA levels in conjunction with high NEFA levels may have depressed dry matter intakes, decreased immune function, decreased pregnancy rates, increased risk of displaced abomasum, and development of fatty liver and ketosis, along with additional ailments.

[0117] FIG. 14 depicts a schematic of a reaction that may be used within the exemplary assay of the disclosure to detect BHBA. The reaction uses a colorimetric enzymatic reaction. In some embodiments, generally speaking, D-3-hydroxybutyrate in the presence of NAD gets converted to acetoacetate and NADH at a pH of 8.5 by a first enzyme. In some embodiments, the first enzyme may include D-3 -hydroxybutyrate dehydrogenase (“3-HBDH”). At the pH of 8.5, the reaction is favored to form NADH, which may be converted to a color (or change in color) using a second enzyme and 2-(p-iodophenyl)-3(p- nitrophenyl)-5 -phenyl tetrazolium chloride (“INT”). In some embodiments, the second enzyme may include diaphorase. In some embodiments, the color or change in color may be detected at 500 nm.

[0118] As the exemplary assay of the disclosure used to detect BHBA within a sample is used within the context of the rotor-based system, the exemplary assay may use one or more beads. In some embodiments, the one or more beads may include reagent beads formed from one or more reagents used within the reaction detailed in FIG. 14 For example, in some embodiments, the reagent bead may include one or more reagents including one or more buffers, one or more sugars, one or more salts, one or more poly ether compounds, one or more reducing agents, one or more enzymes, one or more detergents, one or more coenzymes, one or more surfactants, and/or one or more blocking agents.

[0119] In some embodiments, the one or more buffers, may include tris base, tris hydrocholoride, or the like. In some embodiments, the one or more sugars may include trehalose including D(+) Trahalose dihydrate, dextran, D-mannitol, or the like. In some embodiments, the one or more salts may include sodium oxalate and/or indonitrotetrazolium chloride. In some embodiments, the one or more blocking agents may include bovine serum albumin (BSA). In some embodiments, the one or more poly ether compounds may include polyethylene glycol including PEG-3400. In some embodiments, the one or more enzymes may include 3-Hydroxybutyrate dehydrogenase, and/or diaphorase. In some embodiments, the one or more coenzymes may include NAD. In some embodiments, the one or more surfactants may include n-Octylglucoside.

[0120] In some embodiments, the one or more reagent beads of the disclosure used within the exemplary assay to detect BHBA within a sample may include the formulations disclosed within Tables 2-3.

[0121] Table 2: Exemplary formulation of reagent bead for BHBA assay

[0122] Table 3: Exemplary formulation of reagent bead for BHBA assay

Combination of Exemplary Assays

[0123] In some embodiments, the exemplary assays of the disclosure may be used in combination to provide an assessment of a subject. For example, during times of elevated NEFA uptake within a subject (e.g., an animal), some NEFA are converted back to triglyceride and stored in the liver or are incompletely oxidized to ketones such as BHBA. Subjects (e.g., animals) with high BHBA and non-esterified fatty acids (NEFA) may have depressed dry matter intakes, decreased immune function, decreased pregnancy rates, increased risk of displaced abomasum, and development of fatty liver and ketosis among other ailments. SAA is an inflammatory marker to diagnose if inflammatory diseases need to be investigated in cattle. In some embodiments, in a rotor-based system, one or more of the exemplary assays may be contained within the reaction chambers. For example, in some embodiments, the rotor-based system may include three reaction chambers including the exemplary NEFA assay of the disclosure, the exemplary SAA assay of the disclosure, and the exemplary BHBA assay of the disclosure, with each of the respective assays being disposed within a separate reaction chamber.

[0124] Also disclosed herein are one or more exemplary apparatuses for detecting animal health. In some embodiments, the exemplary apparatus for detecting animal health may include an exemplary apparatus for detecting bovine health. In some embodiment, the exemplary apparatus for detecting bovine health may include a first assay formulated to detect NEFA in a biological sample obtained from a bovine animal in a single incubation step with two or more distinct reagents beads and one or more additional assays. For example, in some embodiments, the apparatus may include a rotor-based system and the one or more additional assays may include additional assays that are configured to detect SAA and BHBA, each of the assays as described above. In some embodiments, the first assay may be contained within a first reaction chamber including a first reaction cuvette. In some embodiments, each additional assay may be contained within an independent reaction chamber or reaction cuvette. For example, a first additional assay may be contained within a second reaction chamber, and a second additional assay may be contained within a third reaction chamber. In some embodiments, the one or more additional assays may include turbidimetric immunoassays, colorimetric enzymatic reactions, or the like.

[0125] In some embodiments, the SAA assay may include a reagent bead that includes a first capture biomolecule coupled to a first particle in a range of about 0.01% by volume to about 0.1% by volume of the SAA assay, and a second capture biomolecule coupled to a second particle in a range of about 0.01% by volume to about 0.1% by volume of the SAA assay. The first capture biomolecule may be configured to bind to a first epitope of SAA, and the second capture biomolecule may be configured to bind to a second epitope of SAA. In some embodiments, the first particle and the second particle of the SAA assay may include latex beads. In some embodiments, the first particle and the second particle of the SAA assay may be configured to aggregate upon binding to the first and second epitopes of SAA, with the aggregation of the first and second particles configured to cause a change in optical absorbance of the SAA assay. In some embodiments, the change in optical absorbance within the SAA assay may be indicative of a concentration of SAA within the biological sample. In some embodiments, the SAA assay may be formulated to detect SAA in sample in a range from about 25 mg/L to about 200 mg/L, inclusive (e.g., about 25, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 mg/L, inclusive)

[0126] In some embodiments, the BHBA assay may include a reagent bead that includes Diaphorase in a range of about 10 U/mL to about 50 U/mL, inclusive, NAD in a range of about 1 mmol/L to about 30 mmol/L, inclusive, , D-3 -hydroxybutyrate dehydrogenase (3- HBDH) in a range of about 3 U/ml to about 300 U/ml, inclusive, and 2-(p-iodophenyl)- 3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) in a range of about 1 mmol/L to about 5 mmol/L, inclusive.

Exemplary Methods

[0127] Disclosed herein are methods for detecting an unknown concentration of one or more analyte within a sample. FIG. 22 depicts a flow chart of a method 2200 for detecting an unknown concentration of one or more analytes within a sample using one or more exemplary assays of the disclosure. In some embodiments, the method 2200 includes mixing a sample with at least a first reagent bead and optionally a second reagent bead in a single incubation step 2202. In some embodiments, mixing the sample with a least a first reagent bead and optionally a second reagent bead in a single incubation step includes mixing the sample with at least a first reagent bead and a second reagent bead using a rotorbased system. In some embodiments, mixing the sample with at least a first reagent bead and optionally a second reagent bead in a single incubation step includes mixing the sample with the first reagent beads and a second reagent bead within a reaction chamber of the rotor-based system. In some embodiments, the sample includes whole blood or plasma. As described above, the one or more exemplary assays that may be used to detect an unknown concentration of one or more analytes within a sample may include assays to detect NEFA, SAA, and/or BHBA.

[0128] In some embodiments, the method 2200 includes exposing the sample to a light source 2204. In some embodiments, exposing the sample to the light source includes exposing the sample to the light source within the rotor-based system described herein. In some embodiments, exposing the sample to the light sources includes exposing the sample to the light source within the one or more reaction chambers.

[0129] In some embodiments, the method 2200 includes measuring an optical signal from the sample 2206. In some embodiments, measuring the optical signal from the sample includes using the rotor-based system to measure the optical signal. In some embodiments, measuring the optical signal from the sample includes measuring the optical signal from the one or more reaction chamber.

[0130] Also disclosed herein are exemplary methods for detecting a target analyte within a sample, methods for quantifying an unknown quantity of NEFA within a biological sample and diagnosing the risk of metabolic disorder in a bovine animal.

[0131] An exemplary method for detecting a target analyte within a sample may include a method for detecting NEFA within a sample. In some embodiments, the method includes incubating a biological sample including a quantity of NEFA with a reagent, the reagent including a first component, a second component, and a third component. In some embodiments, incubating a biological sample including NEFA with a reagent may include incubating within a first reaction chamber of a rotor-based system. [0132] The method includes allowing the first component to interact with the quantity of NEFA to generate a first reaction product, allowing the first reaction product to interact with the second component to generate a second reaction product, and allowing the second reaction product to interact with the third component to generate an indicator signal indicative of the quantity of NEFA. The method also includes interpreting the indicator signal to determine a quantity of the NEFA. Each of the first reaction product, the second reaction product, and the indicator signal are generated in a single incubation operation, as previously described herein.

[0133] As described above, the reagent may include a first reagent bead and a second reagent bead. In some embodiments, the first component may be included within the first reagent bead and the second component may be included within the second reagent bead. As described above, the first component may comprise co-enzyme A, adenosine 5- triphosphate disodium salt (“ATP”), and acyl-coA synthase, and the first reaction product includes Acyl-CoA, AMP, and PPi. As previously described, the second component may include acyl-CoA oxidase, and the second reaction product includes 2,3 -trans-enoyl-CoA and H2O2. As described herein, the third component may include an indicator molecule, 4- aminoantopyrine (4-AAP), and peroxidase. In some embodiments, the indicator molecule may be included in the first reagent bead, while 4-AAP and peroxidase may be included in the second reagent bead. In some embodiments, the indicator molecule may include TBHBA.

[0134] Also disclosed herein is an exemplary method of quantifying an unknown quantity of NEFA within a biological sample. In some embodiments, the method includes mixing a biological sample including a quantity of NEFA with a first reaction bead and a second reaction bead in a solution to form a mixture, the first reaction bead including a first enzyme, a second enzyme, a first small molecule substrate, and a first chromogen, and the second reaction bead including a second chromogen different from the first chromogen, and a third enzyme different from any of the first enzyme, or the second enzyme. The method includes incubating the mixture for a predetermined time at a predetermined temperature, exposing the sample to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum, and measuring an optical signal from the sample, the optical signal indicative of the quantity of NEFA in the biological sample. [0135] As described above, in some embodiments, the first reaction bead further includes a salt (MgCh). In some embodiments, the optical signal may include reflectance, an absorbance spectrum, scattering spectrum, or an emission spectrum. In some embodiments, mixing a biological sample with a first reagent bead and a second reagent bead in a solution to form a mixture and incubating the mixture for a predetermined time at a predetermined temperature may be performed in a reaction cuvette included in a rotor system. In some embodiments, the sample may include whole blood or plasma.

[0136] Also disclosed herein is an exemplary method for diagnosing the risk of metabolic disorder in a bovine animal. In some embodiments, the method includes collecting a biological sample from the bovine animal, assaying the sample for NEFA, wherein the step of assaying comprises three reaction steps of a NEFA assay that occur in a single incubation operation and generate an indicator signal, and measuring an indicator signal to determine a concentration of the NEFA in the biological sample. In some embodiments, the biological sample includes whole blood or plasma. In some embodiments, assaying may be performed in a reaction cuvette included in a rotor system.

[0137] Embodiments and implementations described herein are further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

EXAMPLES

Example 1. Homogenous Single Incubation NEFA Assay

Exemplary Formulation of Reagent Bead #1

[0138] To generate a single homogenous incubation step for the reaction of FIG. 3B used in the exemplary method of detecting NEFA within a sample, a first reaction bead and second reaction bead were generated. The first reaction bead and second reaction bead were formulated according to the Table 4 or Table 5 below using wet chemistry. The first reaction bead and second reaction bead were dropped in liquid nitrogen at a defined rate to generate homogenous beads. The first reaction bead and second reaction bead were lyophilized to remove moisture from the first and second beads. The first reaction bead and second reaction bead were configured to be used in a single well to attain a single homogenous incubation step. This was configured by removing the NEM component, reducing the concentration of CoA, and using the chromogen TBHBA. The exemplary formulation of the reagent bead #1 is as follows:

Table 4: Exemplary Formulation of Reagent Bead #1

Exemplary Formulation of Reagent Bead #2

[0139] The exemplary formulation of reagent bead #2 is as follows:

Table 5: Exemplary Formulation of Reagent Bead #2

Testing the Exemplary NEFA Assay

[0140] Once the reagent beads #1 and #2 were generated according to the formulations above, the exemplary assay was tested for kinetics.

[0141] FIG. 4A depicts a graph of the measured absorbance with respect to time with increasing NEFA concentrations. As seen in the graph of FIG. 4A, the signal intensity increased with corresponding increases in NEFA concentration.

[0142] To determine and confirm specificity of the exemplary NEFA assay, a study was performed testing contrived and real subject samples, calibrated with the reference method compared to the exemplary NEFA method. Each result was plotted on the graph of FIG. 5A. The R² value was 0.97, indicating a high correlation between the reference method and the exemplary method of the disclosure.

[0143] To test the precision of the exemplary NEFA assay, a NEFA precision study was done. The results are demonstrated in FIG. 5B. At different NEFA concentrations across the dynamic range of the assay, the coefficient of variation for each reading is recorded, showing the precision of the exemplary assay.

[0144] A common occurrence within assays that detect one or more target analytes is interference. Interference can disrupt the sensitivity and/or specificity of the assay. Various interference assays were performed to detect interference within the performance of the exemplary assay of the disclosure.

[0145] FIGS. 6-8 depict graphs of measured icteric interference, hemolysis interference, or lipemic interference respectively of the exemplary NEFA assay.

[0146] To detect icteric interference within the NEFA assay, samples were spiked with increasing concentrations of bilirubin and then measured at a specific fixed NEFA concentration near a medical decision point. A medical decision point may include one or more analyte values which are useful in reaching a diagnosis. The one or more analyte values used within the medical decision point may be determined by a consensus of medical professionals and demonstrated by clinical research. The results were plotted on the graph in FIG. 6 and analyzed to understand and determine the impact of increasing bilirubin on the exemplary NEFA assay and at what concentration of bilirubin there was an impact to the NEFA assay. As demonstrated in FIG. 6, the NEFA assay appeared to not interfere with ICT index values below 6, wherein the ICT index values were equivalent to mg/dL.

[0147] To detect hemolysis interference within the NEFA assay, samples were spiked with a concentrated HEME stock and measured at a specific concentration near the medical decision point. The samples were analyzed, and the results were plotted on the graph in FIG.7 and analyzed to understand and determine the impact of increasing hemolysis on the exemplary NEFA assay and at what concentration of hemolysis there was an impact to the NEFA assay. As demonstrated in FIG. 7, the NEFA assay appeared to not interfere with hemolysis (“HEM”) index values below 250, wherein HEM index values were equivalent to mg/dL.

[0148] To detect lipemic interference within the NEFA assay, intralipid was added to samples to simulate a high lipid environment. For example, intralipid was spiked into the samples at a specific concentration of sample near the medical decision point and measured. The results were plotted on the graph in FIG. 8. and analyzed to understand and determine the impact of increasing lipid concentration on the exemplary NEFA assay and at what concentration of lipids there was an impact to the NEFA assay. As seen in FIG. 8, the NEFA assay appears to not interfere with lipid (“LIP”) index values below 2000.

[0149] Many assays may be assembled in one location at a first time and then travel to a user in a different location where a user may use it at a second time. To account for differences in temperatures during travel and length of time from when the assay was assembled to when the assay was used, the exemplary assay was tested for accelerated stability using higher and lower concentrations of NEFA. The exemplary assay was tested at different temperatures over a period of about 12 days, wherein each test was plotted on the graph of FIG. 9A or FIG. 9B. For example, the accelerated stability was conducted at Day 0, Day 3 and Day 10 at 35° C and the results of the percentage of recovery in both time points was calculated and plotted in the table of FIG. 9C. Samples were loaded into a rotor which was maintained at 35° C for 0, 7, or 10 days and then the assay was tested on the respective day.

[0150] The linearity and range of the exemplary NEFA assay was tested. Decreasing concentrations of NEFA were included within samples used within the exemplary NEFA assay. The samples were measured, and the results were plotted on the graphs of FIG. 10.

[0151] Additional tests may be performed that determine one or more of: the extended range of the exemplary NEFA assay, interference studies, limits of detection, stability, precision, and/or correlation with existing NEFA assays that have multiple incubation steps. Example 2: Complete Bovine Sequence Based Detection using Serum Amyloid A Assay

Generation of Reagent Bead #1

[0152] To adapt the exemplary assay to detect SAA within a sample to be used within a rotor-based system, a reagent bead was generated. The reagent bead was generated according to the formulation described in Table 6.

Table 6: Exemplary Formulation of Reagent Bead

[0153] The exemplary SAA assay of the disclosure was tested for specificity, as described below.

[0154] In order to conjugate antibody 1 and antibody 2 to the surface of the reagent bead, antibody 1 and antibody 2 were passively adsorbed onto the surface of the latex reagent bead at a neutral pH using the following protocol. An appropriate volume of stock latex solution (0.1 pm of 8% Sulfate Latex Beads) was added to the anti-SAA antibody described above, and a conjugation Buffer comprising 50mM MES at a pH 6.0 to obtain an antibody /latex ratio of 55.35 pg of anti-SAA antibody per 1 mg of latex added. The conjugation buffer was added at a ratio of 42 pL of conjugation buffer per 8 pL of stock latex solution added. The solution comprising the latex solution, the anti-SAA antibody and the conjugation buffer was incubated overnight with shaking at room temperature. The solution was blocked with glycine to a final concentration of 0.1% Glycine and incubated with shaking at room temperature for 30 min. Tween 20 was added to the solution at a final concentration of 0.5% Tween 20 and the solution was centrifuged at approximately 12000 RPM for 1 hour at room temperature. After centrifugation, the supernatant was discarded and the solution was resuspended in appropriate volume using Latex Storage Buffer comprising 0.1 TRIS, 0.1% Tween 20, pH 7.5. This step was repeated for approximately two additional times for a total of 3 centrifugation cycles. After final centrifugation, the supernatant was discarded, and the solution was resuspended with an exact volume of Latex Storage Buffer to obtain 1% latex conjugate solution.

[0155] The beads were then used within the SSA assay to detect SAA within the sample.

[0156] To test specificity of the exemplary SAA assay, reagent beads were generated as described above. Samples containing various concentrations of SAA were mixed with the reagent beads within the rotor-based system. Samples were measured and the results were plotted on the graph of FIGs. 13A-E. As shown in FIG. 13A and 13B, the R² value was 0.998 and 0.837, respectively. The values for HEM interference, LIP interference, and ICT interference were 490, 450, and 15, respectively (FIGs. 13C, D, and E).

Example 3: BHBA Assay

[0157] To generate a BHBA assay that may be used within the rotor-based system of the disclosure, one or more reagent beads were generated. The one or more reagent beads may include reagents used within the reaction of FIG. 14.

[0158] The reagent beads were generated according to the formulations described in Table 7 or Table 8 as described below .

Table 7: Exemplary Formulation of Reagent Bead

[0159] Table 8: Exemplary Formulation of Reagent Bead

[0160] In generating the reagent bead of Table 7, a final pH of 8.5 was achieved using Tris HCL for pH adjustment. [0161] In generating the reagent bead of Table 8, TNT was combined with water having a temperature greater than about 100 °F (about 37.7 °C) and less than about 212 °F about 100 °C). The solution was sonicated for about 20 minutes or until the INT was completely dissolved within the solution. n-Octylglucoside was immediately added to the solution.

[0162] To determine enzyme activity within the exemplary BHBA assay, an assay endpoint study was performed using the reagent beads generated according to the formulations described above. Samples were mixed with the reagent beads within the rotor-based system and BHBA levels were measured. The measured results compared to values from a reference method were plotted on the graph in FIG. 15 A, having a R² value was 0.999.

[0163] To determine the kinetics of the exemplary BHBA assay, samples having increased BHBA mM concentrations were mixed with the reagent beads in the rotor-based system and the kinetics were measured and plotted on the graph of FIG. 15B.

[0164] To determine and confirm specificity of the exemplary BHBA assay, a correlation study was performed testing real subject samples calibrated with the reference method compared to the exemplary BHBA method. Each result was plotted on the graph of FIG. 16. The R² value was 0.927, indicating a high correlation between the reference method and the exemplary method of the disclosure. This indicates that the exemplary method of the disclosure is as specific as the reference method.

[0165] As described above with the NEFA assay, to account for differences in temperatures during travel and length of time from when the assay was assembled to when the assay was used, the exemplary assay was tested for accelerated stability using higher and lower concentrations of BHBA. The exemplary assay was tested at different temperatures over a period of about 10 days. Samples were loaded into a rotor which was maintained at 35° C for 0, 3 or 10 days and then the assay was tested on the respective day.

[0166] The percent recovery was measured, including at Day 3 wherein the results are plotted on the graph of FIG. 17, and Day 10 wherein the results are plotted on the graph of FIG. 17B. Samples comprising 0.31 mM BHBA demonstrated an increase in % recovery of BHBA, while samples comprising 6.19 mM BHBA demonstrated a slight reduction in % recovery of BHBA, although the % recovery was still in the mid to high 90%. This suggests that the exemplary BHBA still maintained specificity even after 10 days at 35° C.

[0167] To detect hemolysis interference within the BHBA assay, samples were spiked with a concentrated HEME stock and measured at a specific concentration near the medical decision point. The samples were analyzed, and the results were plotted on the graph in FIG.18 and analyzed to understand and determine the impact of increasing hemolysis on the exemplary BHBA assay and at what concentration of hemolysis there was an impact to the BHBA assay. As seen in FIG. 18, the BHBA assay appears to not interfere with hemolysis (“HEM”) index values below 1120.

[0168] To detect lipemic interference within the BHBA assay, intralipid was added to samples to simulate a high lipid environment. For example, intralipid was spiked into the samples at a specific concentration of sample near the medical decision point and measured. The results were plotted on the graph in FIG. 19. and analyzed to understand and determine the impact of increasing lipid concentration on the exemplary BHBA assay and at what concentration of lipids there was an impact to the BHBA assay. As seen in FIG. 19, the BHBA assay appears to not interfere with lipid (“LIP”) index values below 1820.

[0169] To detect icteric interference within the BHBA assay, samples were spiked with bilirubin and then measured at a specific concentration near the medical decision point. The results were plotted on the graph in FIG. 20 and analyzed to understand and determine the impact of increasing bilirubin on the exemplary BHBA assay and at what concentration of bilirubin there was an impact to the BHBA assay. As seen in FIG. 20, the BHBA assay appears to not interfere with icteric interference (“ICT”) index values below 30.

[0170] The linearity of the exemplary BHBA assay was tested. Increasing concentrations of BHBA were included within samples used within the exemplary BHBA assay. The samples were measured, and the results were plotted on the graphs of FIG. 15. The R² value was 0.999.

[0171] Various concepts may be embodied as one or more methods, of which at least one example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. Put differently, it is to be understood that such features may not necessarily be limited to a particular order of execution, but rather, any number of threads, processes, services, servers, and/or the like that may execute serially, asynchronously, concurrently, in parallel, simultaneously, synchronously, and/or the like in a manner consistent with the disclosure. As such, some of these features may be mutually contradictory, in that they cannot be simultaneously present in a single embodiment. Similarly, some features are applicable to one aspect of the innovations, and inapplicable to others.

[0172] In addition, the disclosure may include other innovations not presently described. Applicant reserves all rights in such innovations, including the right to embodiment such innovations, file additional applications, continuations, continuations-in-part, divisionals, and/or the like thereof. As such, it should be understood that advantages, embodiments, examples, functional, features, logical, operational, organizational, structural, topological, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the embodiments or limitations on equivalents to the embodiments. Depending on the particular desires and/or characteristics of an individual and/or enterprise user, database configuration and/or relational model, data type, data transmission and/or network framework, syntax structure, and/or the like, various embodiments of the technology disclosed herein may be implemented in a manner that enables a great deal of flexibility and customization as described herein.

[0173] While specific embodiments of the present disclosure have been outlined above, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the embodiments set forth herein are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the disclosure. Where methods and steps described above indicate certain events occurring in a certain order, those of ordinary skill in the art having the benefit of this disclosure would recognize that the ordering of certain steps may be modified, and such modification are in accordance with the variations of the invention. Additionally, certain of the steps may be performed concurrently in a parallel process, when possible, as well as performed sequentially as described above. The embodiments have been particularly shown and described, but it will be understood that various changes in form and details may be made.

Claims

1. A method, comprising: incubating a biological sample including a quantity of non-esterified fatty acids (“NEFA”) with a reagent, the reagent including a first component, a second component, and a third component; allowing the first component to interact with the quantity of NEFA to generate a first reaction product; allowing the first reaction product to interact with the second component to generate a second reaction product; allowing the second reaction product to interact with the third component to generate an indicator signal indicative of the quantity of NEFA; and interpreting the indicator signal to determine a quantity of the NEFA, wherein the first reaction product, the second reaction product, and the indicator signal are generated in a single incubation operation.

2. The method of claim 1, wherein the reagent comprises a first reagent bead and a second reagent bead.

3. The method of claim 1, wherein: the first component comprises co-enzyme A, adenosine 5-triphosphate disodium salt (“ATP”), and a acyl-coA synthase; and the first reaction product includes Acyl-CoA, adenosine monophosphate, and phosphoric acid (“PPi”).

4. The method of claim 3, wherein the first component is included in the first reagent bead.

5. The method of claim 2, wherein: the second component includes acyl-CoA oxidase; and the second reaction product includes 2,3 -trans-enoyl-CoA and H2O2.

6. The method of claim 5, wherein the second component is included in the second reagent bead.

7. The method of claim 2, wherein the third component includes an indicator molecule, 4-aminoantopyrine (4-AAP), and peroxidase.

8. The method of claim 7, wherein the indicator molecule is included in the first reagent bead, and the 4-AAP and peroxidase are included in the second reagent bead.

9. The method of claim 7, wherein the indicator molecule includes 2,4,6,-tribromo-3- hydroxybenzoic acid (“TBHBA”).

10. The method of claim 2, wherein the incubating is performed in a first reaction cuvette included in a rotor system.

11. A method, comprising:

(a) mixing a biological sample including a quantity of non-esterified fatty acids (“NEFA”) with a first reagent bead and a second reagent bead in a solution to form a mixture, the first reagent bead comprising a first enzyme, a second enzyme, a small molecule substrate, and a first chromogen and the second reagent bead comprises comprising a second chromogen different from the first chromogen, and a third enzyme different from the first enzyme and the second enzyme,

(b) incubating the mixture for a predetermined time at a predetermined temperature;

(c) exposing the sample to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum; and

(d) measuring an optical signal from the sample, the optical signal indicative of the quantity of NEFA in the biological sample.

12. The method of claim 11, wherein the first reagent bead further includes a salt.

13. The method of claim 12, wherein the salt includes MgCh.

14. The method of claim 11, wherein the optical signal is reflectance, an absorbance spectrum, scattering spectrum, or an emission spectrum.

15. The method of claim 11, wherein at least steps (a) and (b) are performed in a reaction cuvette included in a rotor system.

16. The method of claim 11, wherein the sample is whole blood or plasma.

17. A method for diagnosing the risk of metabolic disorder in a bovine animal, comprising: collecting a biological sample from the bovine animal; assaying the sample for non-esterified fatty acids (“NEFA”), wherein the step of assaying comprises three reaction steps of a NEFA assay that occur in a single incubation operation and generate an indicator signal; and measuring an indicator signal to determine a concentration of the NEFA in the biological sample.

18. The method of claim 17, wherein the biological sample includes whole blood or plasma.

19. The method of claim 17, wherein the steps of assaying are performed in a reaction cuvette included in a rotor system.

20. An assay configured to detect non-esterified fatty acids (NEFA) within a sample acquired from a bovine animal, comprising: a composition comprising: a first reagent bead comprising a first enzyme, a second enzyme, and a small molecule substrate, and a first chromogen; and a second reagent bead comprising a second chromogen different from the first chromogen, and a third enzyme different from the first enzyme, and the second enzyme, the first bead and second bead configured to be mixed with the sample in a single incubation step.

21. The assay of claim 20, wherein the assay occurs at a pH between 6.0 and 8.5

22. The assay of claim 20, wherein the sample is whole blood.

23. The assay of claim 20, wherein the sample is plasma.

24. The assay of claim 20, wherein a total assay volume is about 100 pL.

25. The assay of claim 20, wherein the first chromogen is 2,4,6-Tribromo-3- hydroxybenzoic acid (“TBHBA”).

26. The assay of claim 25, wherein the second chromogen is 4-amino-antipyrine (“4- AAP”).

27. The assay of claim 20, wherein the single incubation step is a homogenous incubation step.

28. The assay of claim 20, wherein the small molecule substrate is Coenzyme A and is present at a concentration in a range of about 0. 1 mM to about 0.5 mM.

29. The assay of claim 28, wherein the first enzyme is Acyl-CoA synthetase and is present at a concentration in a range of about 1.5 U/mL to about 2.5 U/mL.

30. The assay of claim 29, wherein the second enzyme is Acyl-CoA oxidase and is present at a concentration in a range of about 6.0 U/mL to about 9.0 U/mL.

31. The assay of claim 30, wherein the third enzyme is perioxidase and is present at a concentration in a range of about 20 U/mL to about 30 U/mL.

32. The assay of claim 20, wherein each of the first reagent bead and second reagent bead further include one or more cryoprotectants.

33. The assay of claim 32, wherein the one or more cryoprotectants is a surfactant.

34. The assay of claim 20, wherein the first reagent bead or the second reagent bead is lyophilized.

35. An apparatus for detecting bovine health, comprising: a first assay formulated to detect non-esterified fatty acids (“NEFA”) in a biological sample obtained from a bovine animal in a single incubation step with two or more distinct reagent beads; and one or more additional assays.

36. The apparatus of claim 35, wherein the apparatus is a rotor system.

37. The apparatus of claim 35, wherein the one or more additional assays are configured to detect serum amyloid A (“SAA”) and/or configured to detect P- Hydroxybutyric acid (“BHBA”).

38. The apparatus of claim 37, wherein the SAA assay includes a reagent bead, the reagent bead including: a first capture biomolecule coupled to a first particle, the first capture biomolecule configured to bind to a first epitope of SSA; and a second capture biomolecule coupled to a second particle, the second capture biomolecule configured to bind to a second epitope of SSA, the first epitope of SAA being different from the second epitope of SAA.

39. The apparatus of claim 38 wherein: the first particle and the second particle include latex beads, the first and second particles configured to aggregate upon binding to the first and second epitopes of the SSA, respectively; and the aggregation of the first and second particles configured to cause a change in optical absorbance of the SAA assay, the change in optical absorbance indicative of a concentration of SAA in the biological sample.

40. The apparatus of claim 37, wherein the BHBA assay includes one or more reagent beads, the one or more reagent beads including nicotine adenine dinucleotide (NAD) in a concentration of about 1 mmol/L to about 30 mmol/L, D-3 -hydroxybutyrate dehydrogenase (3-HBDH) in a range of about 3 U/ml to about 300 U/ml, Diaphorase in a concentration of about 10 U/mL to about 50 U/mL, and 2-(p-iodophenyl)-3(p-nitrophenyl)- 5-phenyl tetrazolium chloride (INT) in a range of about 1 mmol/L to about 5 mmol/L.

41. The apparatus of claim 35, wherein the first assay is within a first reaction cuvette.

42. The apparatus of claim 35, wherein the one or more additional assays are within one or more separate reaction cuvettes.

43. The apparatus of claim 35, wherein the one or more additional assays include turbidimetric immunoassays or colorimetric enzymatic reactions.